CN110090293B - Method for inhibiting side acylation reaction of polypeptide drug in PLGA microsphere - Google Patents
Method for inhibiting side acylation reaction of polypeptide drug in PLGA microsphere Download PDFInfo
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- CN110090293B CN110090293B CN201910387385.4A CN201910387385A CN110090293B CN 110090293 B CN110090293 B CN 110090293B CN 201910387385 A CN201910387385 A CN 201910387385A CN 110090293 B CN110090293 B CN 110090293B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1611—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
- A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
Abstract
The invention discloses a method for inhibiting side acylation reaction of polypeptide drugs in PLGA microspheres. Aiming at a microsphere preparation method which is the most widely applied method in industrialization, namely an emulsion solvent volatilization method, the method achieves the aim of inhibiting acylation side reactions of polypeptides and carriers for a long time by adding a special divalent metal salt in the process of preparing the multiple emulsion. The divalent metal salt has the following characteristics: 1) Is difficult to dissolve in water; 2) Can absorb hydrogen ions (H) generated by the degradation of the microspheres + ) So as to gradually release divalent metal ions; the divalent metal salt preferably absorbs hydrogen ions (H) generated by the degradation of the microspheres + ) And thereafter, can be gradually converted to the form of the monohydrogen or dihydrogen salt with gradually increasing solubility. The method has the advantages of simple process, low technical requirement and remarkable effect of inhibiting the side reaction of acylation, and is suitable for large-scale industrial production.
Description
Technical Field
The invention relates to the field of polypeptide long-acting sustained-release microsphere preparations, in particular to a method for inhibiting side acylation reaction of a polypeptide drug in PLGA microspheres.
Background
Since the twenty-first century, the development cost of drugs based on novel small molecule compound entities has increased day by day, and statistically, the development cost of a single small molecule chemical drug is about $ 1 to $ 5 million. The development cost of the biomolecular drug is relatively low, and the high selectivity and the high specificity to the therapeutic target point lead the biomolecular drug to show great advantages in the treatment of serious diseases. With the rapid development of modern biotechnology, such as DNA recombination technology, more and more biological products are developed and applied clinically. The biomacromolecule drugs on the market are mainly polypeptide drugs. However, the polypeptide drugs have large molecular weight and are difficult to be absorbed through the intestinal membranes, and the mode of drug delivery mainly depends on the parenteral administration mode. The dosage forms on the market are mostly injections, which are divided into injections and long-acting microsphere preparations for injection. In the case of injection, due to the characteristics of short half-life period, low bioavailability, easy inactivation by endogenous protease hydrolysis and the like of polypeptide drugs, repeated administration is required in order to maintain stable blood concentration and ensure good treatment effect. However, in the treatment of chronic diseases such as blood diseases (hypertension) and hormone secretion dysfunction (diabetes), the patient has poor medication compliance due to continuous administration, and medication cost is increased. Therefore, long-acting microsphere formulations for injection have become the mainstream of research, and the drug effect of one dose can be maintained for weeks or even months. The carrier for preparing the microspheres is mostly a biodegradable high polymer material. The preparation is characterized in that: with the slow degradation of the carrier, the drug is gradually released to achieve the purpose of long-term treatment.
Polylactic-co-glycolic acid (PLGA) is a degradable biopolymer, which has been approved by the Food and Drug Administration (FDA) for clinical use because of its good biocompatibility. At present, PLGA has wide application in the biomedical industry, for example, under the trade name Dexon ® And Vicryl ® The surgical sutures of (4), plates, nails, pegs, screws, etc. for fixation of bone and soft tissue. The carrier material for microsphere formulations that have been marketed is mostly PLGA, which is capable of protecting polypeptide drugs from proteolytic hydrolysis, such as the earliest marketed leuprorelin microspheres (Lupron) ® Depot), late goserelin (Zoladex) ® ) Octreotide (Sandostatin) ® LAR Depot), exenatide microsphere (Bydureon) ® ) And triamcinolone acetonide acetate microspheres (ZILRETTA) recently marketed TM ) And the like. The release rate of the drug and the degradation speed of the carrier are controlled by the proportion and the molecular weight of polymer monomers, namely lactic acid and glycolic acid.
However, during the long-term drug release process of PLGA-loaded polypeptide drug microspheres, acylation side reactions are likely to occur between the drug and the carrier, specifically, covalent reactions occur between the amino groups on lysine and arginine residues in the polypeptide drug molecules and the amino groups at the ends of the molecules and the carboxyl groups at the ends of the PLGA molecules. This covalent reaction will produce a range of acylated by-products of the polypeptide drug. The affinity of the acylation product generated after the side reaction of the drug by acylation and the therapeutic target is greatly reduced (the drug effect is reduced), which seriously leads to strong toxic effect and induces the immune reaction of the organism.
In the literature, "Peptide acylation by poly (alpha-hydroxy esters)", it is reported that the acidic microenvironment generated by the degradation of the carrier in the neutralized microspheres can inhibit the acylation side reactions to some extent; in the document "Identification of chemically modified peptide from poly (D, L-lactate-co-glycolide) microspheres under in vitro release conditions", it is reported that when the proportion of lactic acid monomer in the polymer carrier used is high (for example, lactic acid: glycolic acid = 85; the reports of the "Effect of N-tertiary mono-PEGylation on biological activity and pharmacologic kinetics" and US8206735B2 disclose that the side reactions of acylation are greatly improved when the active amino groups in the polypeptide drug are modified with polyethylene glycol (PEG to protect the amino groups). But the effect of inhibiting acylation side reaction caused by neutralizing acidic microenvironment is poor; PLGA using only a high proportion of lactic acid monomer greatly limits the range of carriers that can be selected; the activity of the drug is greatly reduced (the drug effect is reduced) while the amino group is protected by PEGylation. Therefore, a method which is simple in process, suitable for industrial production and capable of effectively inhibiting the acylation side reaction of the polypeptide drugs in the PLGA microspheres is urgently needed to be developed.
In recent years, the documents "A new class of anions of Peptide Acylation and Acylation in PLGA", "Inhibition of Peptide Acylation in PLGA Microspheres with Water-soluble differentiation Salts" and "Minimizing Acylation of peptides in PLGA Microspheres" and the method disclosed in US 9675675 B2, which can fundamentally solve the problem of side reactions of Acylation of polypeptide drugs in PLGA Microspheres, have attracted extensive attention. It is confirmed that soluble acidic oligomer generated in the degradation process of the microsphere is an important substrate for acylation side reaction. Under physiological environment (pH 7.4), amino protonation in polypeptide drug generates amino positive ion (-NH) 3 + ) The acidic oligomer generated by PLGA degradation is ionized to generate carboxyl anion (-COO) - ) The amino positive ions and carboxyl negative ions with opposite charges generate electrostatic adsorption and generate acylation side reactions. The electrostatic adsorption is a precondition for the side reaction of the PLGA and the medicament during acylation. The study found that divalent Metal ion (Mn) 2+ ，Ca 2+ ，Mg 2+ And Zn 2+ Etc.) can effectively inhibit electrostatic adsorption between the oligomer and the polypeptide, thereby greatly inhibiting acylation side reactions. For optimum inhibition, divalent metal salts of high water solubility (e.g., mnCl) are generally selected 2 ，CaCl 2 ，MgCl 2 ，ZnCl 2 Etc.). However, there are two important technical barriers to this strategy: 1) In the process of preparing microspheres by an emulsification method, inorganic salt with high water solubility is easy to diffuse into an external water phase, so that the encapsulation rate of the inorganic salt is low (20 to 30%); 2) At the initial stage of releasing the medicine from the microsphere, the water-soluble inorganic salt is quickly dissolved to form mutually connected aqueous pore channels, and the metal ions are quickly released into the medium from the aqueous pore channels in the microsphere along the osmotic gradient. The low encapsulation efficiency of the divalent metal salt causes insufficient reserve capacity for inhibiting acylation side reactions for a long time and reduced inhibition effect, and the rapid loss of metal ions at the initial stage of drug release greatly reduces the inhibition effect at the middle and later stages. There is no disclosure of methods to improve the encapsulation of divalent metal salts in microspheres and the slow release of metal ions therein.
Disclosure of Invention
The invention discloses a method for intelligently releasing divalent metal ions with high encapsulation and quick response based on the self degradation characteristic of PLGA microspheres. The method mainly aims at the most widely applied microsphere preparation method in modern industrial production, namely an emulsion solvent volatilization method, and achieves the purpose of intelligently releasing divalent metal ions to inhibit side reactions of polypeptide and carrier acylation by adding a special divalent metal salt in the preparation process of the multiple emulsion. The method has the advantages of simple process, low technical requirement and remarkable effect of inhibiting the side reaction of acylation, and is suitable for large-scale industrial production.
The technical scheme adopted by the invention is as follows:
a method for inhibiting the side acylation reaction of polypeptide drugs in PLGA microspheres is characterized in that a special divalent metal salt is added in the process of preparing PLGA polypeptide drug-loaded microspheres by an emulsification method, and gradually releases divalent metal ions to inhibit the side acylation reaction in the microspheres along with the slow degradation of the microspheres, wherein the divalent metal salt has the following characteristics:
1) Is insoluble in water;
2) Can absorb hydrogen ions (H) generated by the degradation of the microspheres + ) So as to gradually release divalent metal ions;
the divalent metal salt is preferably selected to absorb hydrogen ions (H) generated by degradation of the microspheres + ) The latter can be gradually converted into the higher and higher solubility monohydrogen or dihydrogen salt form, and the inhibition effect is better.
The cation of the divalent metal salt is Ca 2+ 、Mn 2+ 、Mg 2+ 、Zn 2+ 、Sr 2+ 、Ni 2+ One of (1); the anion is: CO 2 3 2- 、PO 4 3- 、SO 4 2- 、PO 3 2- And SO 3 2- One kind of (1).
And grinding the divalent metal salt solid particles, sieving, and selecting particles with the particle size of less than 1 micron as an additive.
The divalent metal salt is added to the PLGA oil phase solution.
The mole ratio of the divalent metal salt to the added medicine is as follows: 1 to 10.
The design principle is as follows: in the drug release process of the PLGA drug-loaded microspheres for several months, the carrier material is slowly degraded to gradually generate acidic oligomers and acidic monomer molecules such as lactic acid, glycolic acid and the like (figure 1). One portion of these acidic oligomer molecules is released into the medium and another portion accumulates inside the microsphere and forms a localized acidic microenvironment (fig. 2 shows the release of acidic oligomer into the medium causing a change in pH of the release medium and fig. 3 shows the change in pH inside the microsphere). The acidic oligomer remained in the microsphere is an important substrate for the acylation side reaction of the polypeptide drug. The acidity of the microenvironment represents the concentration of accumulated oligomers and is an important indicator and signal of the severity of the acylation side reactions. The invention utilizes the acidic microenvironment accompanied with the oligomer as a stimulus to stimulate a special divalent metal salt to release divalent metal ions in response according to acidity (intensity degree of acylation side reaction) so as to inhibit the acylation side reaction. One of the divalent metal salts, ca 3 (PO 4 ) 2 For example, the following steps are carried out:
1) During the preparation of the microspheres, due to Ca 3 (PO 4 ) 2 Insoluble, solid form of Ca 3 (PO 4 ) 2 The amount of migration into the external aqueous phase is small and therefore the encapsulation efficiency is high (about 95%). The high encapsulation isThe premise and guarantee of inhibiting acylation side reaction for a long time. And Ca is not degraded in the process of the microspheres (under the condition of no acid stimulation) 3 (PO 4 ) 2 Is solid (insoluble in water) and does not cause unnecessary loss of calcium ions. At this time, most of the calcium is stored in the microspheres in the form of solid, ca 3 (PO 4 ) 2 As a potential calcium ion reservoir.
2) As the microspheres gradually degrade in the release medium, more and more acidic oligomers are produced, ca in solid form 3 (PO 4 ) 2 Will absorb H of the microenvironment + And gradually converted into CaHPO with higher and higher solubility 4 And Ca (H) 2 PO 4 ) 2 (FIG. 4), the acylation side reaction is inhibited by spontaneously supplying a corresponding amount of calcium ion depending on the intensity of the acylation side reaction. The more acidic oligomer is produced, the more vigorous the acylation side reaction, and the more sufficient the amount of calcium ion released from the calcium ion reservoir. The results show that the invention has good effect of inhibiting acylation side reaction (taking octreotide acetate as an example, figure 5, example 1).
The method is realized by the following steps:
the invention adopts water-in-oil-in-water (W) 1 /O/W 2 ) The polypeptide medicine microsphere is prepared through emulsion solvent volatilizing process, and bivalent metal salt as additive is added. The concrete components are as follows:
1) Internal water phase:
medicine preparation: 10% ~100% (w/v, g/ml)
2) Oil phase:
PLGA：5%~100%（w/v, g/ml）
divalent metal salt: 0.5% ~5% (w/v, g/ml)
Surfactant (B): 0.1% -10% (w/v, g/ml)
3) External water phase:
surfactant (B): 0.1% -10% (w/v, g/ml)
The preparation method comprises the following steps:
adding the polypeptide drug into the aqueous solution, dissolving with ultrasound to prepare an internal aqueous phase solution (W) 1 ）。
Adding PLGA into an organic solvent, dissolving by ultrasonic wave, preparing an oil phase solution (O), and adding the ground divalent metal salt particles into the oil phase solution (O).
Mixing the inner water phase drug solution (W) 1 ) Dropwise adding into the oil phase solution (O), and homogenizing and emulsifying to obtain a primary emulsion solution (W) 1 /O）。
Adding a surfactant solution containing a small amount of stable colostrum into the colostrum solution, and performing vortex oscillation to uniformly mix.
Adding said mixed colostrum to a large volume of aqueous external aqueous solution (W) 2 ) In the preparation method, a multiple emulsion solution (W) is prepared by stirring 1 /O/W 2 ）。
And after the organic solvent is completely volatilized, collecting the microspheres, repeatedly washing the microspheres with deionized water, removing supernatant, and freeze-drying the remaining microspheres to obtain the PLGA drug-loaded microspheres.
The polypeptide provided by the invention is directed at polypeptide drugs containing amino groups, including octreotide, exenatide, liraglutide, lanreotide, teriparatide and the like.
The divalent metal salt is characterized by comprising the following components in parts by weight:
1) Are inherently poorly soluble in water;
2) Can absorb hydrogen ions (H) generated by the degradation of the microspheres + ) So as to gradually release divalent metal ions;
3) If the selected divalent metal salt absorbs hydrogen ions (H) generated by the degradation of the microspheres + ) The latter can be gradually converted into the higher and higher solubility monohydrogen or dihydrogen salt form, and the inhibition effect is better.
The divalent metal salt may be, for example: the cation being Ca 2+ 、Mn 2+ 、Mg 2+ 、Zn 2+ 、Sr 2+ 、Ni 2+ One of (1); the anion is: CO 2 3 2- 、PO 4 3- 、SO 4 2- 、PO 3 2- And SO 3 2- To (3) is provided. Grinding the solid particles of the divalent metal salt, sieving, and selectingParticles with a particle size of less than 1 micron are used as additives. Vortex for 5 minutes to disperse uniformly in the organic phase. The cation of the preferred divalent metal salt is Ca 2+ 、Zn 2+ The anion is one of: CO 2 3 2- 、PO 4 3- 、PO 3 2- And SO 3 2- Wherein the particle size of the divalent metal salt particles is less than 0.1 micron.
The molar ratio of the divalent metal salt to the added medicine is as follows: 1; the preferred molar ratio is 1 to 1.
The internal water phase solvent of the invention is: one or more of water, methanol and acetonitrile, wherein the volume of an internal water phase is 50 to 500 mu l, and the concentration of the medicine is 10 to 100 percent (w/v, g/ml). The preferable solvent is one or two of water and methanol, the volume of an internal aqueous phase is 100 to 200 mu l, and the concentration of the medicine is 20 to 50 percent (w/v, g/ml).
The oil phase solvent of the invention is: one or more of dichloromethane, ethyl acetate, acetone, trichloromethane, methyl acetate and tetrahydrofuran, wherein the volume of the oil phase is 1 to 10 ml, the concentration of PLGA is 5 to 100 percent (w/v, g/ml), and the amount of the divalent metal salt is 0.5 to 5 percent (w/v, g/ml). The preferable solvents are dichloromethane and ethyl acetate, the volume of the oil phase is 2 to 5 ml, the amount of PLGA is 20 to 50 percent (w/v, g/ml), and the amount of the divalent metal salt is 2 to 4 percent (w/v, g/ml).
The colostrum is prepared at the homogenizing speed of 10000 to 30000 rpm and the homogenizing time of 0.5 to 5 minutes. The preferred rotation speed is 20000 rpm and the homogenization time is 1 minute.
The surfactant solution for stabilizing the colostrum is a polyvinyl alcohol solution with the volume of 2-10 ml and the concentration of 0.1-10% (w/v, g/ml). Preferably, the polyvinyl alcohol has a concentration of 2 to 5% (w/v, g/ml) and a volume of 2 to 5 ml. The mixed emulsion was vortexed on a vortexer at maximum vortexing speed for 30 seconds to thoroughly mix the emulsion.
The external water phase solution is 0.1 to 10 percent (w/v, g/ml) of polyvinyl alcohol aqueous solution, and the volume of the external water phase solution is 50 to 500 ml, preferably 0.3 to 1 percent (w/v, g/ml) of polyvinyl alcohol aqueous solution, and the volume is 80 to 150 ml.
The stirring speed in the preparation of the multiple emulsion is 100 to 1000 rpm, and the preferred rotating speed is 200 to 500 rpm.
After 6 hours, the organic solvent is completely volatilized, the obtained microsphere solution is centrifuged to remove the supernatant, then the microspheres are repeatedly washed by deionized water, and the supernatant is centrifuged to be removed. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres.
The microspheres prepared by the water-in-oil-in-water emulsion solvent volatilization method have uniform particle size and smooth surfaces (figure 6). The added divalent metal salt has good inhibition effect on acylation side reaction in the microsphere. The process is simple and is suitable for large-scale industrial production.
Has the advantages that: according to the invention, a special divalent metal salt is added in the preparation process of the multiple emulsion, so that the purpose of intelligently releasing divalent metal ions to inhibit the side reaction of acylation of the polypeptide and the carrier is achieved.
Drawings
FIG. 1 is a graph of the degradation of PLGA to produce acidic oligomers.
FIG. 2 is a graph showing the release of acidic oligomers from microspheres into a medium to cause a change in pH of the medium.
FIG. 3 is a graph showing the change in pH inside the microspheres.
FIG. 4 shows a sparingly soluble divalent metal salt Ca 3 (PO 4 ) 2 Gradually converted into a monohydrogen CaHPO with gradually increased water solubility under acidic conditions 4 And the dihydrogen salt Ca (H) 2 PO 4 ) 2 Form diagram.
FIG. 5 shows that 40 mg Ca is added into microspheres containing 20 mg octreotide acetate 3 (PO 4 ) 2 Effect of suppressing side reaction of acylation (example 1).
FIG. 6 shows the addition of Ca to microspheres 3 (PO 4 ) 2 Scanning electron microscopy of (a).
FIG. 7 shows that 50 mg Ca is added into microspheres containing 50 mg octreotide acetate 3 (PO 4 ) 2 Inhibition of acylationEffect of side reaction (example 2).
FIG. 8 shows that 40 mg ZnCO is added into microspheres containing 20 mg octreotide acetate 3 Effect of suppressing side reaction of acylation (example 3).
FIG. 9 shows that 30 mg Ca is added to microspheres containing 30 mg exenatide 3 (PO 4 ) 2 Effect of suppressing side reaction of acylation (example 4).
FIG. 10 shows the addition of 20 mg CaCO to microspheres containing 20 mg octreotide acetate 3 Effect of suppressing side reaction of acylation (example 5).
FIG. 11 shows that 30 mg Ca was added to microspheres containing 50 mg of exenatide 3 (PO 3 ) 2 Effect of suppressing side reaction of acylation (example 6).
FIG. 12 shows that 80 mg Ca is added into microspheres containing 50 mg octreotide acetate 3 (PO 4 ) 2 Effect of suppressing side reaction of acylation (example 7).
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. The following examples are only given to aid the understanding of the method of the invention and its core idea. It should be noted that, for a person skilled in the art, it is possible to make several modifications and improvements to the present invention without departing from the principle of the present invention, and these modifications and improvements are also within the scope of the present invention as defined in the appended claims.
Example 1
Weighing 20 mg of octreotide acetate, and dissolving in 100 mul of deionized water; 600 mg PLGA 503H was dissolved in 2 ml dichloromethane. Weighing Ca after grinding and sieving 3 (PO 4 ) 2 40 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point, 100. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (20000 rpm) for 1 minute. 2 ml of a 2% (w/v, g/ml) solution of polyvinyl alcohol was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). Adding the mixed emulsion to 100ml of a water solution containing 0.5% (w/v, g/ml) of polyvinyl alcohol, and stirred at 350 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The drug release results for 35 days showed that the acylation side reaction rate decreased 85.30% (fig. 5).
Example 2
Weighing 50 mg of octreotide acetate, and dissolving in 200 mul of deionized water; 600 mg PLGA 503H was dissolved in 2 ml dichloromethane. Weighing the ground and sieved Ca 3 (PO 4 ) 2 50 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point 200. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (20000 rpm) for 1 minute. 2 ml of a 2% (w/v, g/ml) solution of polyvinyl alcohol was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). The mixed emulsion was added to 100 ml of a water solution containing 0.5% (w/v, g/ml) of polyvinyl alcohol and stirred at 400 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased by 57.97% (fig. 7).
Example 3
Weighing 20 mg of octreotide acetate, and dissolving in 100 mul of deionized water; 800 mg PLGA 504 was dissolved in 4 ml dichloromethane. Weighing ZnCO after grinding and sieving 3 40 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point, 100. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (20000 rpm) for 1 minute. 2 ml of 4% (w/v, g/ml) polyvinyl alcohol solution was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). Adding the mixed emulsion to 100 ml of a water solution containing 0.5% (w/v, g/ml) of polyvinyl alcohol, and stirred at 350 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And (3) pre-freezing the wet microspheres in a cold trap of a freeze dryer for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze-drying chamber, and freeze-drying the wet microspheres for 48 hours to obtain the dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased 46.80% (fig. 8).
Example 4
Weighing 30 mg of exenatide and dissolving in 100 mul of deionized water; 600 mg PLGA 503H was dissolved in 2 ml dichloromethane. Weighing Ca after grinding and sieving 3 (PO 4 ) 2 30 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point, 100. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (20000 rpm) for 1 minute. To the colostrum 3 ml of a 2% (w/v, g/ml) solution of polyvinyl alcohol was added and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). The mixed emulsion was added to 200 ml of a water solution containing 0.5% (w/v, g/ml) of polyvinyl alcohol and stirred at 300 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased 77.98% (fig. 9).
Example 5
Weighing 20 mg of octreotide acetate, and dissolving in 100 mul of methanol; 1000 mg PLGA 752H was dissolved in 3 ml dichloromethane. Weighing ground and sieved CaCO 3 20 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point, 100. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (15000 rpm) for 1 minute. 2 ml of 2% (w/v, g/ml) polyvinyl alcohol solution was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). Adding the mixed emulsion to 100 ml of a water solution containing 0.5% (w/v, g/ml) of polyvinyl alcohol, and stirred at 350 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased by 48.93% (fig. 10).
Example 6
Weighing 50 mg of exenatide and dissolving in 200 mul of deionized water; 700 mg PLGA 503H was dissolved in 2 ml dichloromethane. Weighing Ca after grinding and sieving 3 (PO 3 ) 2 30 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point 200. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (18000 rpm) for 1 minute. 2 ml of 2% (w/v, g/ml) polyvinyl alcohol solution was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed). The mixed emulsion was added to 100 ml of a water solution containing 0.8% (w/v, g/ml) of polyvinyl alcohol and stirred at 400 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased by 54.53% (fig. 11).
Example 7
Weighing 50 mg of octreotide acetate, and dissolving in 100 mul of deionized water; 600 mg PLGA 503H was dissolved in 2 ml dichloromethane. Weighing the ground and sieved Ca 3 (PO 4 ) 2 80 mg was added to the organic phase and vortexed to suspend the organic phase uniformly. At this point, 100. Mu.l of the drug solution was immediately added dropwise to the organic phase and homogenized on a homogenizer (13000 rpm) for 1 minute. 3 ml of 3% (w/v, g/ml) polyvinyl alcohol solution was added to the colostrum and vortexed on a vortex shaker for 30 seconds (maximum vortex speed).The mixed emulsion was added to 200 ml of a water solution containing 0.3% (w/v, g/ml) of polyvinyl alcohol and stirred at 350 rpm for 6 hours. When the organic solvent is completely volatilized, centrifuging the obtained microsphere solution to remove the supernatant, then repeatedly washing the microspheres with deionized water, and centrifuging to remove the supernatant. And putting the wet microspheres into a cold trap of a freeze dryer for pre-freezing for 6 hours, taking out the wet microspheres and putting the wet microspheres into a freeze drying chamber, and freeze-drying for 48 hours to obtain dry microspheres. The 35-day drug release results indicated that the acylation side reaction rate decreased by 72.96% (fig. 12).
Claims (7)
1. A method for inhibiting the side acylation reaction of polypeptide drugs in PLGA microspheres is characterized in that a special divalent metal salt is added in the process of preparing PLGA polypeptide drug-loaded microspheres by an emulsification method, and gradually releases divalent metal ions along with slow degradation of the microspheres to inhibit the side acylation reaction in the microspheres, wherein the divalent metal salt has the following characteristics:
1) Is difficult to dissolve in water;
2) Can absorb hydrogen ions (H) generated by the degradation of the microspheres + ) So as to gradually release divalent metal ions;
the divalent metal salt absorbs hydrogen ions (H) generated by the degradation of the microspheres + ) Thereafter, can be gradually converted to the form of the monohydrogen or dihydrogen salt with gradually increasing solubility;
grinding and sieving the divalent metal salt solid particles, selecting particles with the particle size less than 1 micron as an additive, and adding the divalent metal salt into the PLGA oil phase solution;
the mole ratio of the divalent metal salt to the added medicine is as follows: 1, 1 to 10;
the cation of the divalent metal salt is Ca 2+ 、Zn 2+ One of (1); the anion is: CO 2 3 2- 、PO 4 3- One kind of (1).
2. The method for inhibiting the side effect of the acylation of the polypeptide drugs in the PLGA microspheres according to claim 1, wherein the polypeptide drugs comprise octreotide, exenatide, liraglutide, lanreotide, teriparatide, etc.
3. The method for inhibiting the side reaction of the acylation of the polypeptide drugs in the PLGA microspheres according to claim 1, comprising the steps of:
1) Adding the polypeptide drug into the aqueous solution, dissolving with ultrasound, and preparing the internal water phase solution W 1 ；
2) Adding PLGA into an organic solvent, dissolving by ultrasonic waves, preparing an oil phase solution O, and adding divalent metal salt particles into the oil phase solution O;
3) Mixing the inner water phase drug solution W 1 Dropwise adding into the oil phase solution O, and homogenizing and emulsifying to obtain a primary emulsion solution W 1 /O；
4) Adding a surfactant solution containing a small amount of stable colostrum into the colostrum solution, and performing vortex oscillation at the maximum vortex speed for 30 seconds to uniformly mix the colostrum solution and the surfactant solution;
5) Adding the mixed colostrum into the large-volume external water phase aqueous solution W 2 In the preparation method, the compound emulsion solution W is prepared by stirring 1 /O/W 2 ；
6) And after the organic solvent is completely volatilized, collecting the microspheres, repeatedly washing the microspheres with deionized water, removing supernatant, and freeze-drying the remaining microspheres to obtain the PLGA drug-loaded microspheres.
4. The method for inhibiting the side acylation reaction of the polypeptide drugs in the PLGA microspheres according to claim 3, wherein the internal aqueous phase solvent is one or more of water, methanol and acetonitrile; the oil phase solvent is one or more of dichloromethane, ethyl acetate, acetone, trichloromethane, methyl acetate and tetrahydrofuran; the external water phase solution is 0.3 to 1 percent (w/v, g/ml) of polyvinyl alcohol aqueous solution.
5. The method for inhibiting the side effect of polypeptide drug acylation in PLGA microspheres according to claim 3, wherein the cation of the divalent metal salt is Ca 2+ 、Zn 2+ The anion is one of: CO 2 3 2- 、PO 4 3- In the above-mentioned manner, the first and second substrates are,the particle size of the divalent metal salt particles is less than 0.1 micron.
6. The method for inhibiting the side acylation reaction of the polypeptide drugs in the PLGA microspheres according to claim 3, wherein the drug concentration in the inner water phase is 20% to 50% (w/v, g/ml), the PLGA concentration in the oil phase is 20% to 50% (w/v, g/ml), and the amount of the divalent metal salt in the oil phase is 0.5% to 5% (w/v, g/ml).
7. The method for inhibiting the side acylation reaction of the polypeptide drugs in the PLGA microspheres according to claim 3, wherein the homogenization speed for the colostrum preparation is 10000 to 30000 rpm, and the homogenization time is 0.5 to 5 minutes.
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